(1) Field of the Invention
This invention relates generally to trophic or growth factors and, more particularly, to chimeric GDNF family growth factors which activate GFRα1-RET but do not substantially activate GFRα2-RET or GFRα3-RET, growth factors derived therefrom and methods therefor.
(2) Description of the Related Art
The development and maintenance of tissues in complex organisms requires precise control over the processes of cell proliferation, differentiation, survival and function. A major mechanism whereby these processes are controlled is through the actions of polypeptides known as “growth factors”. These structurally diverse molecules act through specific cell surface receptors to produce these actions.
Growth factors termed “neurotrophic factors” promote differentiation, maintain a mature phenotype and provide trophic support, promoting growth, function and survival of neurons. Neurotrophic factors reside in the nervous system or in innervated tissues. Nerve growth factor (NGF) was the first neurotrophic factor to be identified and characterized (Levi-Montalcini Montalcini et al., J. Exp. Zool. 116: 321, 1951). NGF exists as a non-covalently bound homodimer that promotes the survival and growth of sympathetic, neural crest-derived sensory, and basal forebrain cholinergic neurons. Other effects of NGF, including effects on non-neuronal cells of the endocrine and immune systems (including inflammatory cells) are disclosed in, e.g., Levi-Montalcini and Booker, Proc Nat'l Acad Sci 46: 384-391, 1960; Johnson et al. Science 210: 916-918, 1980; Crowley et al., Cell 76: 1001-12, 1994; Snider and Johnson, Ann Neurol 26: 489-506, 1989; Hefti, J Neurobiol 25: 1418-35, 1994; Scully and Otten, Cell Biol Int 19: 459-469, 1995; Otten and Gadient, Int. J. Devl Neurosci 13: 147-151, 1995; and Horigome et al. J Biol Chem 269: 2695-2707, 1994.
In recent years it has become apparent that growth factors fall into classes, i.e. families or superfamilies based upon the similarities in their amino acid sequences. These families include, for example, the fibroblast growth factor family, the neurotrophin family and the transforming growth factor-beta (TGF-β) family. As an example of family member sequence similarities, TGF-β family members have 7 canonical framework cysteine residues which identify members of this superfamily.
The NGF family is the prototype of such a family of growth factors. Brain-derived neurotrophic factor, the second member of this family to be discovered, was shown to be related to NGF by virtue of the conservation of all six cysteines that form the three internal disulfides of the NGF monomer (Barde, Prog Growth Factor Res 2: 237-248, 1990 and Liebrock et al. Nature 341: 149-152, 1989). By utilizing the information provided by brain-derived neurotrophic factor of the highly conserved portions of two factors, additional members (NT-3, NT-4/5) of this neurotrophin family were rapidly found by several groups. (Klein, FASEB J 8: 738-44, 1994).
Recently, a new family of neurotrophic factors has been identified whose members are not structurally related to NGF and other neurotrophins but are structurally similar to TGF-β. As described in U.S. Pat. No. 5,739,307, and U.S. patent application Ser. Nos. 08/931,858 and 09/220,531, the known members of this subfamily of the TGF-β superfamily include glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin, and artemin. The placement of GDNF, neurturin, persephin, and artemin into the same growth factor family, also referred to as the GDNF ligand family, is based on the similarities of their physical structures and biological activities. For example, human persephin has about 40% sequence identity and about 43% sequence conservation with human GDNF; about 49% sequence identity and about 50% sequence conservation with human neurturin; and about 45% sequence identity and about 48% sequence conservation with human artemin. In addition, these four proteins have the seven cysteine residues typical of TGF-β family members.
The GDNF family ligands support the survival of dopaminergic ventral midbrain neurons cultured from the embryo. Additionally, GDNF, neurturin, and persephin support the survival of spinal and facial motor neurons in both in vitro survival and in vivo injury paradigms, identifying these ligands as potential therapeutic agents in the treatment of neurodegenerative diseases (Henderson et al., Science 266: 1062-1064, 1994; Horger et al., J Neurosci. 18: 4929-37, 1998; Klein et al., Nature 387: 717-721, 1997; Lin et al., Science 260: 1130-1132, 1993; Milbrandt et al., Neuron 20: 245-53, 1998; Oppenheim et al., Nature 373: 344-346, 1995), reviewed by Grondin and Gash, J Neurol. 245(11 Suppl 3): 35-42, 1998). However, whereas GDNF and neurturin both support the survival of peripheral sympathetic, parasympathetic, sensory, and enteric neurons (Buj-Bello et al., Neuron 15: 821-828, 1995; Ebendal et al., J Neurosci Res 40: 276-284, 1995; Heuckeroth et al., Dev Biol 200: 116-29, 1998; Kotzbauer et al., Nature 384: 467-470, 1996; Trupp et al., J of Cell Biology 130: 137-148, 1995), persephin does not support survival in any peripheral neurons tested to date (Milbrandt et al. supra).
The GDNF family ligands share receptors and signal transduction pathways (Creedon et al., Proc. Natl. Acad. Sci. USA 94: 7018-7023, 1997; Durbec et al., Nature 381: 789-793, 1996; Trupp et al., Nature 381: 785-789, 1996; Baloh et al., Neuron 18: 793-802, 1997). These proteins act through a multicomponent receptor complex in which a transmembrane signal transducing component, the Ret protein-tyrosine kinase (RET), is activated upon the binding of a growth factor of the GDNF family with a member of a family of closely related co-receptors named GFRα. A characteristic feature of the GFRα co-receptor family is that its members have no transmembrane domain and are attached to the cell surface via a glycosyl-phosphatidylinositol (GPI) linkage (Durbec et al., Nature 381: 789-793, 1996; Jing et al., Cell 85: 1113-1124, 1996; Treanor et al., Nature 382: 80-83, 1996; Trupp et al., Nature 381: 785-789, 1996; Baloh et al., 1997, supra). The members of the GFRα family include GFRα1 (previously known as GDNFRα, TmR1 and RetL1), GFRα2 (previously TmR2, NTNRα and RetL2), GFRα3 (previously TmR3) (GFRα Nomenclature Committee, Neuron 19: 485, 1997) and possibly GFRα4, a receptor currently only identified in the chicken (cGFRα4) (Enokido et al., Current Biology 8: 1019-1022, 1998).
Results from extensive in vitro and in vivo experimentation has established that for each GDNF family ligand there is a preferred GFRα receptor, to which the GDNF family ligand binds with highest affinity and most potently activates RET. These preferred interactions are GDNF-GFRα1, neurturin-GFRα2, and artemin-GFRα3 (Baloh et al., 1997, supra; Baloh et al., Proc. Natl. Acad. Sci., USA 95: 5801-5806, 1998; Jing et al., 1996, supra; Jing et al., J Biol. Chem. 272: 33111-33117, 1997; Klein et al., Nature 387: 717-721, 1997; Treanor et al., 1996, supra). Persephin does not bind or activate any of the known mammalian GFRα's but does bind to chicken GFRα4 (Milbrandt et al., supra; Baloh et al., 1998, supra, Enokido et al. supra). However, despite these preferred interactions, there is also clear cross-talk between the different ligand-receptor pairs. The known alternative interactions are neurturin-GFRAα1, artemin-GFRα1, and GDNF-GFRα2 (Baloh et al., 1998, supra; Sanicola et al., Proc. Natl. Acad. Sci., USA 94: 6238-6243, 1997; Suvanto et al., Hum. Molec. Genet. 6: 1267-1273, 1997). Thus, there is no known naturally occurring GFRα1-RET activating GDNF family ligand which does not also activate another GFRα-RET complex.
Some information is available about the roles of each GFRα receptor. Recent analysis of GFRα1-deficient mice indicated that GFRα1 is the only physiologically critical GDNF receptor in kidney organogenesis and enteric nervous system development (Cacalano et al., Neuron 21: 53-62, 1998; Enomoto et al., Neuron 21: 317-324, 1998). However, GDNF-deficient mice have greater losses in peripheral ganglia than GFRα1-deficient mice, suggesting that GDNF can utilize other receptors to support survival of peripheral neurons, likely GFRα2-RET (Cacalano et al., supra; Enomoto et al., supra). Nevertheless, several lines of evidence argue that the effects of GDNF, neurturin and artemin on dopaminergic ventral midbrain neurons are mediated through the GFRα1-RET receptor system. First, since GFRα3 is not expressed in the ventral midbrain, and artemin cannot utilize GFRα2, survival promotion of these neurons by artemin is likely through its ability to activate GFRα1-RET (Baloh et al., supra). Second, GFRα2 expression is diffuse and weak in the pars compacta region of the substantia nigra, and does not colocalize with tyrosine hydroxylase staining (TH) neurons, in contrast to the significantly stronger expression of GFRα1, which does colocalize with TH staining neurons (Horger et al., J. Neurosci 18: 4929-4937, 1998). Finally, the ability of both GDNF and neurturin to support the survival of dopaminergic ventral midbrain neurons is lost in GFRα1 knockout mice, indicating that at least in the embryo the survival promotion of dopaminergic ventral midbrain neurons is only through GFRα1-RET signaling (Cacalano et al., 1998).
While the in vitro interactions between the different GNDF family ligands and GFRα's is now relatively well understood, the molecular basis of this specificity and cross-talk has been heretofore unknown. The crystal structure of GDNF reveals that it is a disulfide-bonded dimer that is significantly similar to the structure of TGF-β2, as predicted by the cysteine spacing of its primary sequence (Daopin et al., Science 257: 369-373, 1992; Eigenbrot and Gerber, Nat. Struct. Biol. 4: 435438, 1997; Schlungger and Grutter, Nature 358: 430434, 1992). However, the structure itself yields only speculative information regarding receptor-binding surfaces. Furthermore, analogy to other TGF-β superfamily members regarding receptor-binding surfaces would likely be unfounded as the receptors used by GDNF and the TGF-β's are drastically different and likely to have little, if any, structural similarity.
It is now generally believed that neurotrophic factors regulate many aspects of neuronal function, including survival and development in fetal life, and structural integrity, function and plasticity in adulthood. Since both acute nervous system injuries as well as chronic neurodegenerative diseases are characterized by structural damage and, possibly, by disease-induced apoptosis, it is likely that neurotrophic factors play some role in these afflictions. Indeed, a considerable body of evidence suggests that neurotrophic factors may be valuable therapeutic agents for treatment of these neurodegenerative conditions, which are perhaps the most socially and economically destructive diseases now afflicting our society. For example, GDNF has been shown to relieve disease symptoms in several animal models of Parkinson's disease (reviewed by Grondin and Gash, supra). Nevertheless, because there is clear cross-talk between the different ligands and receptors, it would be desirable to have growth factors of the GDNF family which are selective for particular receptors. In particular, because there are several central and peripheral sites of GFRα2-RET or GFRα3-RET expression which could lead to side effects as a result of treatment of central nervous system injury or neurodegenerative diseases with GDNF, neurturin or artemin, there is a need for GDNF family ligand members which are more specific in activating GFRα1-RET. The identification of a GDNF family ligand which only activates GFRα1-RET, would also lead to a further elucidation of the relative roles of each GFRα receptor.